Elevated plasma level of the glycolysis byproduct methylglyoxal on admission is an independent biomarker of mortality in ICU COVID-19 patients

Biomarkers to identify ICU COVID-19 patients at high risk for mortality are urgently needed for therapeutic care and management. Here we found plasma levels of the glycolysis byproduct methylglyoxal (MG) were 4.4-fold higher in ICU patients upon admission that later died (n = 33), and 1.7-fold higher in ICU patients that survived (n = 32),compared to uninfected controls (n = 30). The increased MG in patients that died correlated inversely with the levels of the MG-degrading enzyme glyoxalase-1 (r2 = − 0.50), and its co-factor glutathione (r2 = − 0.63), and positively with monocytes (r2 = 0.29). The inflammation markers, SSAO (r2 = 0.52), TNF-α (r2 = 0.41), IL-1β (r2 = 0.25), CRP (r2 = 0.26) also correlated positively with MG. Logistic regression analysis provides evidence of a significant relationship between the elevated MG upon admission into ICU and death (P < 0.0001), with 42% of the death variability explained. From these data we conclude that elevated plasma MG on admission is a novel independent biomarker that predicts mortality in ICU COVID-19 patients.

www.nature.com/scientificreports/ ferences in the mean age and weights of uninfected DM patients and infected patients. Uninfected individuals without DM were slightly younger and weighed less than ICU patients. There were twice as many males than females in this cohort of patients. A higher percentage of non-DM patients were on antiviral regimens than DM patients (41% vs 12%). Similar percentages of non-DM than DM patients were on antibiotics and steroids. Comorbidities are also shown in Table 1. Of the thirty-four non-DM patients, sixteen died (47%), and seventeen of thirty-one DM patients died (55%). The blood analyte profile of patients used in this study are shown in Table 2. They were divided into five groups: individual tests, blood cell/coagulation parameters, renal function parameters, liver function parameters and lipid profiles. Blood glucose levels in ICU COVID-19 patients without DM were higher than that of un-infected individuals. Neutrophils were higher, and lymphocytes basophils and eosinophils were lower in ICU COVID-19 patients. There were no changes in monocytes levels between non-infected controls and ICU COVID-19 patients. Ferritin was also elevated, and hemoglobin and hematocrit were lower in ICU COVID-19 patients. There were no significant differences in Na + , K + , Cl − , Ca 2+ , phosphorous, and Mg 2+ levels between uninfected and ICU COVID-19 patients. However, albumin levels were lower, and urea was higher. Liver enzymes were also higher in ICU COVID-19 patients.

Immune cells in ICU COVID-19 patients. Since upregulation of glycolysis in immune cells is required
to orchestrate their highly specific series of responses to clear viral infection and repair cellular damage, we measured immune cells levels in ICU patients. Neutrophils were significantly higher, and lymphocytes, basophils, and eosinophils were significantly lower in ICU COVID-19 patients compared to uninfected individuals (Fig. 3A,B,D,E, respectively). No significant difference was found in the amounts of monocytes in ICU COVID- Plasma levels of MG, glutathione, Glo1, SSAO, TNF-α, IL-1β, CRP in non-DM, uninfected DM and ICU COVID-19 patients. Studies have shown that patients with DM are at higher risk for severe COVID-19 outcomes compared with non-DM patients [11][12][13] . In this study, MG was 2.7-fold higher (1326 μg/ml HSA-MG in uninfected DM vs 495 μg/ml HSA-MG in uninfected control, P < 0.05), and glutathione was 2.3-fold (342 nmol/ml uninfected DM vs 777 nmol/ml in uninfected control, P < 0.05) lower in uninfected DM patients compared to uninfected non-DM patients (Fig. 5A,C). However, there was no significant difference in Glo1 levels between uninfected non-DM and uninfected DM patients (45 ng/ml uninfected DM vs 42 ng/ml in uninfected control) (Fig. 5E). MG was also significantly higher in COVID-19 DM ICU patients compared to unin-  (Fig. 5A). There were no significant differences in plasma levels of MG, glutathione, Glo1, and CRP between non-DM and DM patients upon ICU admittance (Figs. 5A,C,E, 6G). However, SSAO, TNF-α and IL-1β were significantly higher in plasma of DM ICU patients than non-DM ICU patients (Fig. 6A,C,E).  www.nature.com/scientificreports/ When non-DM and DM ICU patients were further subdivided into those that survived and died, plasma MG was significantly higher, and glutathione and Glo1 were significantly lower in non-DM and DM that died compared to ICU patients that survived (Fig. 5B,D,F). There were no differences between SSAO, TNF-α, and IL-1β levels between non-DM and DM patients that survived and died (Fig. 6B,D,F). CRP was higher in DM ICU patients that died than in DM patients that survived (Fig. 6H).
Plasma levels of neutrophils, lymphocytes, monocytes, basophils and eosinophils in non-DM, uninfected DM and ICU COVID-19 patients. Next, we investigated whether neutrophils lymphocytes, monocytes basophils, and eosinophils levels are altered in non-DM and DM patients that survived and died. Compared to uninfected non-DM patients, the amount of neutrophil was higher, and the amounts of lymphocytes and eosinophils were lower in both non-DM and DM ICU patients, but not monocytes (Figs. 7A,C and 8C). Basophils levels were also significantly lower in DM but not in non-DM ICU patients compared to uninfected non-DM patients (Fig. 8A). In this study, there were also no significant differences in the amounts of neutrophils, lymphocytes, monocytes basophils and eosinophils between non-DM and DM COVID-19 patients (Figs. 7A,C,E, 8A,C). When non-DM and DM ICU patients were further subdivided into those that survived and died, we found higher levels of neutrophils in DM ICU patients that died compared to those that survived (Fig. 7B), and higher levels in monocytes in both non-DM and DM that died compared to non-DM and DM that survived (Fig. 7F). There were no significant differences in the number of lymphocytes and basophils in non-DM and DM COVID-19 ICU patients that died and survived (Figs. 7D, 8B) There was trend towards more eosinophils DM ICU patients that died compared to DM ICU patients that survived, but the data was not significant (P > 0.05, Fig. 8D).
Mortality in ICU COVID-19 patients with low and high plasma MG. Plasma MG levels were used to further separate ICU patients that survived into those with low MG (up to twofold higher than that in uninfected non-DM) and moderate MG (2-threefold higher than uninfected non-DM). MG level in uninfected non-DM 495 μg/ml HSA-MG, "□") was used as the reference. For comparison, we also included MG levels in uninfected DM patients, "⋄" (Fig. 9A). All patients with low plasma MG upon admittance (11/65, 17%), were discharged from ICU. The median time to discharge was 7 days, range of 5-22 days. Patients (21/65, 32%) with moderate MG levels that survived had a mean time to discharge of 8.5 days, with a range of 3-32 days.
Patients that died were also divided into two group. The first group contained patients with MG less than fourfold than uninfected controls (moderate MG patients), and the second group contained patients with MG > fourfold than uninfected controls (high MG patients), Fig. 9A. In all patients with moderate MG that died, median time to death (13/65, 20%) was 25 days, range 14-31 days, and in the second group (20/65, 31%), the median time to death was 14 days with a range of 9-26 days. Figure 9B-D shows Kaplan-Meier curves for all patients, non-DM patients that died, and DM that died, respectively. These data show that the higher the plasma MG, the earlier the onset of death, regardless of whether the patient had DM or not.
A forward selection logistic regression model indicates evidence of a significant relationship between MG and COVID-19 patients (chi-square = 24.90, df = 1, P < 0.0001). Dead patients were found to have significantly higher MG (P < 0.0001) compared to their survived counterparts. The model correctly predicted 67% of death cases and explained 42% of the variability.
Correlations between MG, glutathione, Glo1, SSAO, TNF-α, IL-1β, CRP, age and immune cells in ICU COVID-19 patients that died. Next, we investigated correlations between plasma levels of glutathione, Glo1, age and immune cells with MG levels to gain insights into reasons for MG accumulation, with glutathione, Glo1, age and immune cells as independent variables. Since MG induces inflammation 43,46 Figure 2. Plasma levels of MG, glutathione, Glo1, SSAO activity, TNF-α, IL-1β and CRP in uninfected controls and ICU COVID-19 patients that survived and died. (A) MG levels in plasma from ICU COVID-19 patients that survived and died were significantly higher than that in uninfected controls. MG levels in plasma from ICU COVID-19 that died were also significantly higher than ICU COVID-19 patients that survived. (B) Glutathione in plasma from ICU COVID-19 patients that survived and died were significantly lower than that in uninfected controls. Glutathione levels in plasma from ICU COVID-19 that died were also significantly lower than ICU COVID-19 patients that survived. (C) Glo1 in plasma from ICU COVID-19 patients that survived were not significantly different from that in uninfected controls. Glo1 levels in plasma from ICU COVID-19 patients that died were significantly lower than that in uninfected controls and ICU COVID-19 patients that survived. (D) SSAO activities in plasma from ICU COVID-19 patients that survived and died were significantly higher than that in uninfected controls. However, there were no significant difference in plasma SSAO activities between ICU COVID-19 patients that survived and died. (E) TNF-α levels in plasma from ICU COVID-19 patients that survived and died were significantly higher than that in uninfected controls. However, there were no significant difference in plasma levels of TNF-α in ICU COVID-19 patients that survived and died. (F) IL-1β levels in plasma from ICU COVID-19 patients that survived and died were significantly higher than that in uninfected controls. However, there were no significant difference in plasma levels of IL-1β in ICU patients that survived and died. (G) CRP levels in plasma from ICU COVID-19 patients that survived and died were significantly higher than that in uninfected controls. However, there were no significant difference in plasma levels of CRP in ICU patients that survived and died. Data shown are mean ± S.E.M from n = 30 in uninfected controls (26.6% females), n = 33 died (42.4% females) and n = 32 in survived (37.5% females). Statistical significance levels are shown above data points on each graph. www.nature.com/scientificreports/ we also investigated correlations between plasma MG and SSAO, TNF-α, IL-1β, and CRP levels with MG as the independent variable. In this study, strong inverse correlations were found between plasma MG and glutathione (r 2 = − 0.63) and Glo1 (r 2 = − 0.50), Fig. 10A,B. A weaker but significant positive correlation was also found between MG and age of patient (Fig. 10C). Strong positive correlation between plasma MG and SSAO (r 2 = 0.52) and moderate correlation with TNF-α (r 2 = 0.41) were found (Fig. 10D,E). Weaker but significant correlations were also observed with IL-1β (r 2 = 0.25), and CRP (r 2 = 0.26) (Fig. 10F,G). A strong positive correlation was also found between the number of monocytes and plasma MG levels in ICU patients that died (Fig. 11C). There were no significant correlations between MG and neutrophils, lymphocytes, basophils, eosinophils, neutrophil:lymphocyte and neutrophil:monocyte and lymphocyte:monocyte ratios (Fig. 11A,B,D-H).

Discussion
About 10% of people infected with SARS-CoV-2 develop ARDS requiring intensive care hospitalization [3][4][5] . About 50% of COVID-19 ICU patients will also die 50 . To date, biomarkers to predict which ICU COVID-19 patients are at high risk of mortality are insufficient 4,[15][16][17][18][19] . The principal finding of this cross-sectional study is that high plasma levels of the glycolysis byproduct MG upon admission into ICU with ARDS is a predictor of death in COVID-19 patients. This conclusion is based on our findings that mean plasma MG of COVID-19 patients admitted into the ICU that later died was 4.4-fold and 1.8-fold higher than that in uninfected non-DM controls and uninfected DM individuals, respectively. Similar levels of MG have been reported in plasma of uninfected   [52][53][54] . Mean plasma MG in COVID-19 patients on admission into ICU that survived, was 2.5-fold higher than that of uninfected non-DM controls, and not significantly different to that of uninfected DM patients.
Others have shown that glycolysis and the oxidative arm of the PPP are upregulated in SARS-CoV-2-infected cells to provide the substrates needed for replication 55,56 , and in immune cells to eliminate SARS-CoV-2 infection and repair any cellular damage [27][28][29] . As such, we concluded that MG synthesis is likely increasing in COVID-19 patients. In this study, we focused on whether MG degradation is being compromised in COVID-19 ICU patients. We found that plasma levels of glutathione, and Glo1 were significantly lower in ICU COVID-19 patients that died compared to ICU COVID-19 patients that survived. In an earlier report Horowitz et al. 57 showed that oral and intravenous glutathione and the glutathione precursors (N-acetylcysteine) attenuated activation of NF-κB, cytokine storm syndrome and respiratory distress syndrome seen in COVID-19 patients with pneumonia. Thus, it is likely that intravenous glutathione and the glutathione precursors (N-acetylcysteine) administration were alleviating the oxidative stress and providing the glutathione needed for the formation of MG-glutathione hemiacetal. Glutathione is synthesized in two sequential reactions. In the first reaction, γ-glutamylcysteine ligase (GCL; EC 6.3.2.2) converts l-glutamate and l-cysteine into γ-glutamylcysteine, and in the second reaction, glutathione synthetase (GSS; EC 6.3.2.3) adds glycine to γ-glutamylcysteine to form glutathione 38,58,59 . In a recent study Moolamalla et al. 56 found that the modifier and catalytic subunits genes of γ-glutamylcysteine ligase were downregulated in A549 (alveolar epithelial cells derived from lung adenocarcinoma), ACE2-induced A549, and normal human bronchial epithelial (NHBE) cells infected with SARS-CoV-2. These investigators also found downregulation of GSS gene in in A549, ACE2-induced A549, Calu3 (lung epithelial cells derived from lung adenocarcinoma) cells infected with SARS-CoV-2-infected and in lung biopsy from SARS-CoV-2-infected patients. Our findings are consistent with others showing that glutathione synthesis is being compromised in COVID-19 ICU patients. The increase in oxidative stress induced by SARS-CoV-2 infection may also be lowering the amount of reduced glutathione in host cells/tissues. Additionally, we also found a reduction of plasma levels of Glo1 protein in COVID-19 patients that died. Moolamalla et al. 56 reported down regulation of Glo1 gene in ACE2 transduced A549 and Calu3 cells infected with SARS-CoV-2. To date, the underlying cause(s) for the reduction plasma Glo1 levels in plasma of COVID-19 patients that died remain poorly defined. What we know is that the promoter region of human GLO1 has a functionally operative antioxidant response element (ARE) [39][40][41] . Under non-stress conditions, the antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) binds to the ARE region of GLO1 to induce its expression. NF-κB antagonizes the binding of Nrf2 to ARE to inhibit Glo1 expression. Since activation of NF-κB is upregulated in COVID-19 patients 60 , the increase in activated NF-κB could also account in part for the reduction plasma Glo1. HIF-1α also binds to the ARE of GLO1 to suppress Glo1 expression 39,41 . HIF-1α is one of the two subunits of the heterodimeric transcription factor that regulates cellular and systemic adaptive responses to low oxygen (hypoxia) 24,28,30,33 . HIF-α stabilization can also occur under normoxia during immunity and inflammation via upregulation of PI3K, AKT, mTOR, and STAT3 pathways in polarized M1 macrophages 30 . When oxygen delivery is compromised as is the case with respiratory distress syndrome and ischemia in COVID-19, HIF-1α escapes degradation, allowing it to migrate to the nucleus and induce transcription of HIF-1α target genes, including those involved in glycolysis and erythropoiesis 33,61,62 . The increase in glycolysis and suppression of Glo1 expression by HIF-1α inadvertently leads to an increase in MG. To the best of our knowledge, there is no published literature on linking HIF-1α upregulation and elevation in MG in COVID-19 patients. Figure 5. Plasma levels of MG, glutathione, and Glo-1 in ICU COVID-19 patients without diabetes mellitus (non-DM) and with diabetes (DM) and separated into those that survived and those that died. (A) MG levels were significantly higher in non-DM ICU COVID-19 patients compared to uninfected non-DM but not uninfected DM individuals. There was a significant difference in MG levels between DM ICU COVID-19 patients and uninfected non-DM or uninfected DM individuals. There was not a significant difference in MG levels between non-DM ICU COVID-19 and DM ICU COVID-19. (B) Plasma of MG in non-DM and DM ICU COVID-19 patients that died were significantly higher than that survived. There was not a significant difference in MG levels between DM ICU COVID-19 patients that survived and uninfected DM individuals. However, there was a significant difference in MG levels between non-DM ICU COVID-19 patients that died and uninfected DM individuals. (C) Glutathione levels were significantly lower in non-DM and DM ICU COVID-19 patients than that in uninfected non-DM individuals. There were no significant differences in glutathione levels in non-DM and DM ICU COVID-19 patients compared to uninfected DM individuals. (D) Plasma of glutathione levels in non-DM and DM ICU COVID-19 patients that died had significantly lower than that in patients that survived. There was also a significant difference in MG levels of non-DM and DM ICU COVID-19 that died compared to uninfected DM individuals. There was not a significant difference in glutathione levels between non-DM and DM ICU COVID-19 patients that survived and uninfected DM individuals.  www.nature.com/scientificreports/ In ICU patients that died, we also found blood neutrophils were 450% (P < 0.0001) and monocytes were 20% (P < 0.05) higher than that in non-infected controls. Lymphocytes and eosinophils were also 37% and 68% lower than that in non-infected controls, respectively. Basophil levels were not significantly different between uninfected controls and ICU COVID-19 patients. Other have reported neutrophilia, lymphopenia and monocytosis in COVID-19 patients were associated with poor outcomes 63 . While it is clear that high levels of neutrophils and monocytes arise from the body's response to eliminate SARS-CoV-2 31,32,60,64 , specific mechanisms by which neutrophilia and monocytosis contribute to poor outcomes in COVID-19 patients remain poorly understood. We posit that neutrophilia and monocytosis could be contributing to poorer outcomes in COVID-19 patients in part by increasing production of the cytotoxic glycolysis metabolite MG.
In this study, all ICU COVID-19 patients had significantly higher plasma levels of the inflammation markers originating from activation of several pathways; SSAO from increased expression of the inflammation-induced protein vascular adhesion protein-1 48 , TNF-α from activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) 65 , and IL-1β from activation of the inflammasome 66-68 compared to uninfected non-DM individuals. However, when ICU COVID-19 patients were subdivided into those that survived and died, there were no significant differences between SSAO, TNF-α, and IL-1β, suggesting that these inflammation biomarkers are not predictive of death.
Individuals with DM are at increased risk of severe respiratory and adverse outcomes including death following SARS-CoV-2 infection compared to non-DM patients 6,[10][11][12] . This prompted us to separate our ICU cohorts further into non-DM and DM and with subdivision into those that survived and those that died. In this study, the amount of MG in plasma of ICU COVID-19 patients without DM were not significantly different from that in uninfected DM patients. However, ICU COVID-19 patients with DM had significantly higher levels of plasma MG than uninfected DM patients. MG levels in ICU COVID-19 with and without DM patients that died were 70% and 68% higher than that in non-DM and DM that survived, respectively. As in this study, other have also reported decreased glutathione and Glo1 level in DM patients compared to non-DM patients 69,70 . However, in this study, glutathione and Glo1 levels in ICU COVID-19 patients with and without DM that died were significantly lower than that in ICU COVID-19 patients with and without DM that survived and uninfected DM, indicating that the degradation of MG is being compromised in non-DM and DM patients that died.
To gain further insight into the relationship between plasma MG levels and death, the logistic regression analysis revealed evidence of a significant relationship between MG and COVID-19 patients that died. Our model also correctly predicted 67% of death cases in ICU COVID-19 patients and explained 42% of the death variability. Correlational studies were also conducted to investigate the relationships between plasma MG as the dependent variable. We found strong inverse correlations between MG and glutathione, Glo1, and age. As an independent factor, MG also positively correlated with SSAO and TNF-α. These data suggest MG elevation is arising from both an increase in synthesis via glycolysis and from impaired degradation due to reduction in glutathione and Glo1. To the best of our knowledge, these data are the first to show that elevated plasma MG in ICU COVID-19 patients upon admission is predictive of death.
This study is not without limitations. ELISA assays were used for measuring MG (HSA-MG), Glo1, TNFα, and IL-1β. Although the protocols for these assays were provided by the manufacturer and followed as per instruction with appropriate controls, additional work studies are needed using other methodologies, including mass spectrometry, Western blot assays and quantitative polymerase chain reactions (q-PCR) for measurements of MG, Glo1, TNF-α, and IL-1β.
In summary, the present study shows for the first time that elevation in plasma levels of the cytotoxic glycolysis metabolite MG can be used as a novel independent biomarker that predicts mortality in ICU COVID-19 patients. This elevation in MG is arising from increased glycolysis in SARS-CoV-2 infected and immune cells and from impairment in MG degradation due to down regulation of Glo1 and glutathione. Our working hypothesis Figure 6. Plasma SSAO activities and levels of TNF-α, IL-1β and CRP in ICU COVID-19 patients without diabetes mellitus (non-DM) and with diabetes (DM) and separated into those that survived and those that died. (A) SSAO activities in plasma from non-DM and DM ICU COVID-19 patients were significantly higher than uninfected non-DM individuals. There was also a significant difference in plasma SSAO activities between non-DM and DM ICU COVID-19 patients. (B) No significant differences in SSAO activities in plasma from non-DM and DM ICU COVID-19 patients that died and survived. (C) TNF-α levels in plasma from non-DM and DM ICU COVID-19 patients were significantly higher than uninfected non-DM individuals. There was a significant difference in TNF-α in plasma from non-DM and DM ICU COVID-19 patients. (D) No significant differences in plasma levels of TNF-α in non-DM and DM ICU COVID-19 patients that died and survived. (E) IL-1β levels in plasma of non-DM and DM ICU COVID-19 patients were significantly higher than that in uninfected non-DM individuals. IL-1β levels in plasma from ICU COVID-19 with DM were also significantly higher than of ICU-COVID-19 patients without DM.   Fig. 12. Since elevated MG is cytotoxic to cells, we posit that therapeutic strategies to lower MG levels may be useful in reducing adverse clinical outcomes in SARS-CoV-2 infection. These new data suggest that post COVID syndrome may be due in part to vascular and tissue damage initiated by elevated MG levels.

Materials and methods
Study participants. This study was approved by the ethical committee of Imam Abdulrahman Bin Faisal University, Al Kubar (IRB # 2020-05-184), and of Qatif Central Hospital (QCH-SREC0229/2020). COVID-19 patients admitted to the ICU in Qatif Central Hospital between October 2020 to Feb 2021 were recruited into this study after informed consent was taken. All ICU patients had respiratory rate ≥ 30 beats/min; blood oxygen saturation ≤ 93% at rest; arterial oxygen partial pressure (PaO 2 )/oxygen concentration (FiO 2 ) ratio < 300; lung infiltrates > 50% of the lung field within 24-48 h. SARS-CoV-2 infection was confirmed by two sequential realtime reverse transcriptase polymerase chain reaction assays (Abbott Molecular Real Time SARS-CoV-2 assay, Abbott Molecular, Des Plaines, IL, USA) from nasopharyngeal, oropharyngeal and bronchoalveolar lavage fluid swab specimens. Age, medical history, and prior medications were obtained from medical records. The attending physicians reported the outcome of the ICU patients as either dead or discharge. Uninfected volunteers (non-diabetic and diabetic) were recruited from Al-Ahsa and Qatif areas to serve as controls. All assays were performed in accordance with guidelines for handling and assaying blood samples from COVID-19 patients by Imam Abdulrahman Bin Faisal University.